Bioinformatics Analysis Of Key Enzymes In The Synthetic Pathway Of Melatonin In Plants And Callus Induction And Culture Of Transgenic Tobacco With Mytilus Galloprovincialis Foot Protein Type 5(mgfp-5) Gene

Part 1:Melatonin(MT)is a small molecule in the vertebrate pineal secretion of neuroend ocrine hormones,and later found widespread in plants,animals and other organisms.Melatonin can play a role in regulating vertebrate biological rhythm and photoperiod r esponse,improve sleep disorders,enhance immunity and so on;Melatonin can also pr omote plant rooting and improve plant antioxidant capacity.In plant cells of tryptopha n catalyzed by tryptamine 5-hydroxylase(T5H),tryptophan decarboxylase(TDC),serot onin N-acetyltransferase(AANAT),hydroxyindole-O-methyl transferase(HIOMT)four k inds of enzymes,In the metabolic process,melatonin decomposition produces 2-hydrox y melatonin.Studies on the key enzymes involved in the synthesis of melatonin in p1 ants help to understand the mechanism of plant melatonin,dynamic accumulation and its function.Based on this,this study uses a comparative basic group approach to ana lyze and compare the biological information data of TDC,T5H,AANAT and HIOMT in different plant species were analyzed and compared,predict or summarize the basi c properties of their proteins,domains,advanced structures and molecular evolutionary information,provide a reference for the study of key enzymes in melatonin synthesisThe main results achieved are:(1)In addition to the molecular weight of Dioscorea TDC protein,the molecular weight of TDC protein in different plants was mainly distributed between 55000Da-58000Da.The theoretical isoelectric point distribution is between 5.35-6.65.In addition to the Hordeum vulgare TDC1,TDC2 protein and the Dioscorea TDC2 protein,the other TDC proteins were unstable proteins;All of the TDC proteins in the samples w ere hydrophilic and had two distinct hydrophobic regions;In addition to Catharanthus roseus TDC,Rauvolfia verticillata TDC,Ophiorrhiza japonica TDC and rice TDC,All other TDC protein transporters in the samples were found in the cytoplasm;The resu Its of phylogenetic tree analysis showed that the TDC protein was divided into 3 bran ches.(2)In addition to the molecular weight of Morus T5H1 protein,the molecular we ight of T5H protein in different plants was mainly distributed between 50000Da-57000 Da.The theoretical isoelectric point distribution is between 8.80-9.10.In addition to th e Morus T5H1 and T5H2,the other T5H proteins were unstable proteins;All of the T5H proteins in the samples were hydrophilic and had two distinct hydrophobic region s;There is almost no Taxus brevifolia T5H 1-5 protein in chloroplast transit peptide s ignals exist,The scores of mitochondrial transport peptides is higher.The results of p hylogenetic tree analysis showed that the T5H protein was divided into 3 branches.(3)Analysis of AANTA and HIOMT protein in Chlamydomonas shows that amin o acid residues of the protein molecular weight and isoelectric point in HIOMT protei n were higher than in AANAT protein.The AANAT protein is a stable protein in Chl amydomonas reinhardtii and The HIOMT protein is unstable;The entire polypeptide c hain of AANAT protein is hydrophilic and has two distinct hydrophobic regions,the e ntire polypeptide chain of HIOMT protein is hydrophobic,and there are three obvious hydrophilic regions in the whole polypeptide chain.Part 2:Mussel Adhesive Protein(MAP),also named Mussel Foot Protein(Mfp),is secreted by Mytilidae's byssus gland.It has a high adhesiveness in water and corrosion resist ance,at the same time it is non-toxic,non-immunogenic and its biocompatibility is ex cellent biocompatibility.Natural mussel adhesive protein,easy to cure when extracted and low content,difficult to obtain.Now we use genetic engineering technology in th e prokaryotic expression(E.coli)and eukaryotic expression(yeast,tobacco)system to recombine mussel adhesion protein and research its nature and function.In prophase,we expressed the Mgfp-5 protein(Mytilus galloprovincialis protein type 5,Mgfp-5)g ene in tobacco,and obtained the transgenic tobacco.Plant tissue culture has the advan tages of fast cell proliferation,short culture period,uniform material,good reproducibil ity,no seasonal and environmental limitation,there have been many reports on tobacc o tissue and cell culture,gene transformation and secondary production.However,it h as not been reported to study the recombinant protein Mgfp-5 by means of tobacco.E xperiments were using the T1-generation mgfp-5 tobacco from the previously transferre d as an explant,induce callus which can stablely express recombinant protein Mgfp-5.The main results achieved are:(1)In this experiment,we induced the callus of transgenic Ti generation seedings.The induction rate was not less than 90.0%on containing different concentrations of 2,4-D,6-BA and NAA on MS medium with Kanamycin concentration of 20 mg/L.H owever,the induction rate was 100%when 2,4-D was 1.0 mg/L,6-BA was 0.5 mg/L and NAA was 0.1 mg/L.(2)The growth curves of the callus in the solid and liquid culture were all "S"shapes.During 12-27 days of solid culture,the fresh weight growth rate was 3.73,liq uid culture time was shortened by 6 days,the quality of fresh will increase 8.56-8.63 times in the first 6-15 days of time.(3)In the callus of solid and liquid culture,we detected the expected bands by PCR,RT-PCR and Western blot.This shows the stable expression of exogenous gene mgfp-5 and protein Mgfp-5.Mgfp-5 protein stably expressing callus established may provide a new way for the recombinant protein Mgfp-5 raw materials,and it can prov ide a reference for the study of plant expression Mussel Adhesive Proteins.